SUMMARY Parasitic nematodes infect a significant proportion of the world's population and exact an enormous toll of human illness. These parasites infect their hosts as developmentally arrested infective larvae (usually third-stage larvae, (L3i) that resume development once they enter the host. Drawing upon extensive relevant findings in Caenorhabditis elegans, we have uncovered definitive evidence that insulin-like (ILS) and steroid-nuclear hormone receptor (NHR) signaling regulate L3i development before and during the infective process in the parasitic nematode Strongyloides stercoralis. In this renewal application, we propose to complete our studies on regulation of L3i development by ILS in S. stercoralis and initiate new research on the potential of a conserved steroid NHR signaling pathway as a therapeutic target in this parasite. The first of our two Specific Aims asks whether insulin signaling regulates formation and maintenance of L3i by S. stercoralis. Specifically we will determine whether the insulin-like receptor kinase Ss-DAF-2 and the PI3 kinase Ss-AGE-1 block arrest of L3i and promote their developmental reactivation in the host and whether insulin-like peptides Ss-ILP-6 and Ss-ILP-7 promote developmental reactivation and arrest of L3i, respectively. Our approach will involve analyzing phenotypes that result from expressing dominant transgene constructs based on these molecules in S. stercoralis. Specific Aim 2 will ask whether signaling through the Ss-DAF-12 NHR augments regulatory ILS effects in S. stercoralis and whether this small molecule receptor could be a target for chemotherapy based on naturally occurring steroids or their analogs. We propose to identify the natural ligands of Ss-DAF-12 and compare their activity in regulating L3i arrest and reactivation to the DAF-12 ligands from C. elegans, D4- and D7-dafachronic acids (DA). We will use chemical inhibitors to probe endogenous NHR DAF-12 function in S. stercoralis and use a gerbil model of infection to determine whether administered Ss-daf-12 inhibitors or DA can prevent development of L3i, clear adult worm infections and/or block fulminant autoinfection, a potentially fatal complication of human strongyloidiasis. These experiments will directly reflect the potential of DAF-12 NHR function as a chemotherapeutic target in parasitic nematodes.